Effect of RNAi-mediated knockdown of unc-13 and Rbp on gene expression in motor neurons of Drosophila melanogaster third instar larva

Reliable neurotransmission is dependent on coordinating entry of calcium into the terminal boutons of a presynaptic neuron with the exocytosis of vesicles into the synaptic cleft. This function fulfilled by an assembly of proteins in a specialized region of the presynaptic plasma membrane called the active zone (AZ). Here we use the fruit fly neuromuscular junction as a model synapse to test the consequences of specific depletion of two key AZ proteins, unc-13 and RIM-binding protein (Rbp), by RNAi knockdown on the transcriptome of these motor neurons. We find that decreased expression of Rbp results in concomittant decrease of expression in unc-13 and that the sets of differentially expressed genes between the two knockdowns are highly similar. We show that these knockdowns result in increased expression of genes involved in neuropeptide signalling, while reducing expression of other genes encoding the components of the AZ and voltage-gated K+ channels. Taken together, our findings suggest that knockdown of AZ components results in gene expression changes that bias the presynaptic neuron towards increased firing to compensate for the loss of AZs. Overall design: To investigate the consequences of impaired neurotransmission on the transcriptome of motor neurons, we made use of two fruit fly lines, one with Gal4-mediated control of nuclear mCherry driven by the motor neuron-specific OK6 promoter and another with dsRNA constructs against unc-13 or Rbp in the attP2 insertion site. Rbp and unc-13 knockdowns were compared to controls with an empty attP2 site. Motor neurons were isolated from dissected and dissociated brain tissue from each genotype by FACS and each population profiled by RNA-seq.